In the biotechnology industry, there is an ongoing need to develop faster and more economical bioassay systems to test and screen compounds. The current standard format is a microwell or microtiter plate having a dimension approximately 3×5 inches and including 96 wells. Higher density systems using 384-wells are now being incorporated into the research industry. However while the plates comprise more wells, they still have the drawbacks associated with individual plates: the need for storing plates, labeling them and analyzing each plate individually. Often, the tasks associated with such plates are accomplished by hand, including pipetting and syringe dispensing. While this work is tedious and time consuming, when magnified by 384 for each well of the plate, it is even more so. In addition, hand pipetting and syringe dispensing are generally only accurate to 1 microliter (ul) and above.
One problem encountered with sample handling of the prior art is demonstrated by small-volume polymerase chain reaction (PCR) assays. Single nucleotide polymorphisms (SNPs) represent the most abundant type of sequence variation in the human genome and can and should be useful tools for many diverse applications, including delineating the genetic architecture of complex traits and diseases, pharmacogenetics, forensics and evolutionary studies.
Historically, genetic studies have been predicated on identifying and employing genetic variation to address problems of biological significance. An impressive SNP resource already exists as several hundreds of thousands have been deposited into publicly accessible databases, such as the National Center for Biotechnology Information's dbSNP and others. However, without parallel progress in SNP genotyping technology, the use of any database is seriously circumscribed. In addition to SNPs, other allelic variations provide abundant information on genetic variation, population dynamics, genetic mutations and disease diagnostics. Insertion/Deletion (indel) Polymorphisms are a class of polymorphisms based on length differences among nucleotide alleles. The best current estimate is that 20% of all human polymorphisms are of the insertion/deletion (indel) type. Indels can be broken down into a roughly 50:50 mix of multiallelic and diallelic polymorphisms. Multiallelic indels include the minisatellites (also called VNTRs) and the short tandem repeat polymorphisms (STRPs) (also called microsatellites or simple sequence length polymorphisms (SSLPs)). Minisatellites are relatively rare and typically have repeat lengths of a few tens of nucleotides with tandem repeat copy numbers in the hundreds to thousands. STRPs are abundant and have repeat lengths of 1-6 nucleotides with tandem repeat copy numbers, mostly <30. Diallelic indels are also common, but are only just now beginning to be studied in detail (see below). All of the diallelic indels and most of the STRPs have the desirable feature of being able to be analyzed simply by PCR followed by gel electrophoresis. Indels are believed to be an attractive alternative to SNPs.
Novel genotyping methods amenable to high-throughput analysis should ideally be gel-free, robust, inexpensive and simple to perform. To this end, these requirements have inspired the development of a variety of genotyping assays, including the oligonucleotide ligation assay “OLA” (Landegren, U., et al. (1988) Science 241: 1077-80); genetic bit analysis “GBA” (Nikiforov T. T., et al. (1994) Nucleic Acids Res. 11:4167-75); mass spectroscopy (Griffin, T. J., et al. (1999) Proc. Natl. Acad. Sci. 25: 6301-6306), “chip” technology (Wang., et al, supra), TaqMan (Livak, K. J., et al. (1995) PCR Methods Appl. 4:357-62) and dynamic allele specific hybridization “DASH” (Howell, W. M., et al. (1999) Nat. Biotechnology 17:87-88). Although many SNP genotyping methods have been developed, no single technology has emerged as being clearly superior due to limitations such as cost, complexity and accuracy.
Recently, new methods for SNP genotyping, in which the primers are labeled with a fluorophor, have been reported (Myakishev, M. et al. (2001) Genome Res. 11:163-169). This method relies on PCR amplification of genomic DNA with two tailed allele-specific primers that introduce priming sites for universal primers having the fluorescent tag. The fluorophors are selected to emit at different wavelengths and are thus seen as different colors, in this case red and green. The reactions are carried out in a microtiter plate, and following the reaction the plate is read by a fluorescence plate reader. Identification of the emitted color identifies which specific primer annealed to the genomic DNA, and determination of which primer was used indicates which allele is present in the genomic copy. Of note, the authors found that 40 ng of DNA per 20 ul reaction in a 96 well plate was optimal.
Other systems are described in the following patents, published patent applications and references (“references”). While the references attempt to automate or increase the sensitivity of the disclosed inventions, they are limited to specific applications or are limited by large volumes of samples and reactants.
U.S. Pat. No. Re 28,339 to Maxon describes an analysis system for the multiple analysis of a single sample. This system comprises a transfer strip made from an elongated tape having a plurality of liquid samples adsorbed thereto. The tape is used in conjunction with an analyzing apparatus such that each adsorbed aliquot of the sample may be analyzed by a separate system. The disclosure is limited to sample adsorption as a means of retaining the sample on the tape. In addition, since the retention method is adsorption, the detection method is limited to the sample that is adsorbed thereto, not the product of a reaction that occurs within the tape.
U.S. Patent Application 2002/0001546 to Hunter et al. describes methods for screening substances in a microwell array. The method requires loading an array of capillary tubes having dispensing ends, disposing each dispensing end in proximity to a distinct through-hole and transferring the liquids through the through-holes of the platen through the capillary tubes. The method is directed toward a means of filling the wells rather than providing a substitute to the microtiter plates already present in the prior art. In addition, the method described by Hunter et al. is disadvantaged by using relatively large volumes of about 1 ul.
U.S. Pat. No. 3,979,264 to Buerger describes a band for carrying out microbiological examinations. The band resembles a tape or strip having depressions which hold nutrient media or agar. Bacteria are spotted on the media. The tape can be rolled or folded for incubation or storage. The disclosure does not contemplate a means of analysis but, rather, is limited to a means of maintaining organisms in a nutrient media.
U.S. Pat. No. 3,620,678 to Guigan describes a system for multiple automatic analysis. The system includes a tape resembling a roll of film having holes along its side such that the film can be automatically driven by means of a pin or sprocket. The tape is formed from two layers such that there are cells composed within the tape. One facet of the invention includes the automatic filling of the cells with samples to be analyzed. Means of analysis is contemplated to be spectrophotometric, and the detector system is envisioned to be capable of automatically driving the film through the detector for its analysis. However, the samples are only present in a single suspension, not components of a reaction, and the volumes are quite large, about one cubic centimeter.
U.S. Pat. Nos. 6,355,487 and 6,254,297 and U.S. Published Patent Application 2002/0041829 to Kowallis describe a method and apparatus for transferring small volumes of substances. The apparatus comprises a conveyor belt having a plurality of substrates, the substrates being adapted to hold microtubes such that the tubes can be inserted into the substrate and reagents added to the tubes as the conveyor belt moves along. In another embodiment, the conveyor belt itself may be adapted to comprise substrates such that microtubes can be inserted directly into the wells of the conveyor belt. The apparatus does not include a method for analysis, but is envisioned to provide a method for the production of microarrays allowing for the analysis of samples.
U.S. Pat. No. 6,284,546 to Bryning et al. discloses a method and device for photodetection. The device comprises a means of placing a drop of a sample and reagent liquids on a planar support such that the droplets are allowed to mix. The planar support is movable such that the support can be moved through a detector and the emitted light quantified.
U.S. Pat. No. 5,207,986 to Kadota et al. discloses an apparatus for the automatic analysis of biological samples comprising a conveyor belt system for conveying a sample rack, a rack storage unit, an analysis unit and an identification unit such that the analysis unit identifies the samples in the rack.
U.S. Pat. No. 5,092,466 to Anderson describes an apparatus and method for storing samples of protein gene products, cells or DNA. The samples are sealed in packets which are then attached to film. The film can be labeled such that the packets are accurately identified. The invention is to be utilized in the storage and inventory of biological samples, but is not used or contemplated for use as a reservoir for containing a reaction.
U.S. Patent Application 2002/0055179 to Busey et al. describes an apparatus and method for ultra-high-throughput fluorescent screening of samples. The samples are held in a microtiter plate having V-shaped wells. The apparatus comprises at least two light guides such that a light source adjacent to the plate can illuminate an individual well and the emitted light can be guided to an adjacent detector.
U.S. Pat. Nos. 3,923,463 and Re 30,627 to Bagshawe et al. describe an apparatus for performing chemical and biological analysis. The invention describes a method for the handling of large numbers of samples where sample tubes are loaded into racks, the racks loaded into cassettes and the cassettes transferred from station to station for appropriate dilution, reagent addition and analysis.
While these references attempt to provide means to more easily store and analyze samples, they suffer from certain inadequacies: they use relatively large sample volumes; and they represent isolated steps in reacting samples, adding reaction mixtures, analyzing the reaction products and storing those products for further analysis.
There is a need to develop automation on a much smaller scale. As described in R&D Magazine (January 2002, pp A3-A5), companies are now moving to “nanotechnology,” i.e., working in the nanoscale range. There is a definite trend for an assay system that is precise, accurate, efficient, small and economical, yet has a high-throughput.
Companies, such as TOMTEC (Hamden, Conn.), Cartesian Technologies (Irvine, Calif.), Gilson, Inc. (Middleton, Wis.), Molecular Devices Corporation (Sunnyvale, Calif.) and Zymark Corp. (Hopkinson, Mass.), have all moved toward developing smaller, faster systems.
Current technology uses “microwell” or “microtiter” plates having volumes ranging from 1 to 1000 μl to prepare samples and contain reactions. Plates of this nature are injection molded, and the larger well volumes are not suitable for very small volumes in the nanoliter (nl) range. In addition, nanoliter wells may require a special shape in order to position the contents optimally for mixing in the well. Therefore, a more optimally designed well is needed to position and contain the sample.
For high-throughput screening, a continuous process is needed for optimum performance and reduced cost. As may be appreciated, an ability to run large quantities of reactions in very small volumes is limited by at least four factors. First, very low-volume reactions are much more susceptible to operator error in pipetting and transferring of samples and reagents. Second, if an appropriate detector is not available to analyze the reaction products efficiently within their margin of error, small-volume reactions are not worthwhile. Third, if manual manipulations are required, the time needed to process the sample is not affected regardless of the reaction volume. Fourth, a combination of the first three deficiencies limits the overall reproducibility of the analysis. Some laboratories have started to use rail systems to form an assembly line type operation for sample handling with microtiter plates and to minimize manual handling. However, these systems are limited by the constraints of the microtiter plate itself.
TOMTEC is currently developing a MICROTAPE system, which is an endless track of microwells formed on a plastic tape for conducting assays. However, each well is circular in format and requires a relatively large volume (>1 microliter) of reagent to obtain reproducible results. In addition, TOMTEC has a method for sealing the microwell tape comprising heat sealing with a covering tape. One problem with this method is that it requires perforations in the sealing tape to release trapped air, as well as a vacuum device to assure flat juxtaposition of the sealing tape to the microwell tape. Further, use of a microwell tape device for sample screening is hindered by the lack of a detector capable of analyzing the contents of the wells.
Currently, there is no method or device suitable for the continuous, small-volume, high-throughput analysis of tagged biological samples. Commercial units require manual manipulation and volumes in the microliter range. Therefore, there is a need for a continuous feed-through unit to perform sample analysis of a large number of very low-volume reactions without extra handling of the reaction mixtures.